Efficient, rapid, save and cost efficient purification of mammalian IgG antibodies and/or Fab fragments thereof, in particular human and/or humanized IgG antibodies and/or Fab fragments thereof is a much studied problem in the art. With the advent of new antibody based medicaments, purification of IgG and/or Fab fragments thereof becomes a more critical and costly step in the production of antibody based medicaments, requiring a high degree of purity. In addition, such therapeutic antibodies must retain their binding affinity as well as biological activities including effector functions.
For the purification of all four subclasses of human or humanized IgG, i.e. human/humanized IgG1, IgG2, IgG3 and IgG4, commonly used purification methods comprise the use of classical biochemical separation and purification techniques such as anion/kation exchange, size-exclusion/gelfiltration, precipitations and use of specific affinity ligands. Commonly used affinity ligands are bacterially derived Protein-A and Protein-G, monoclonal antibodies and camelid antibodies or binding fragments derived therefrom that bind one or more of the four subclasses of human or humanized IgG.
Protein A binds to the CH2-CH3 interface of human IgG1, human IgG2 and human IgG4 (Fc) and as such cannot be used for the purification of human IgG3 and thus not for all human IgG subclasses. Protein A shows binding to some human VH domains of the VH3 family but not to human VH1 and VH2 (Jansson et al (1998) FEMS Immunology and Medical Microbiology 20:69-78) and therefore Protein A cannot be used as a generic tool for purification of all human IgG derived Fab fragments. Furthermore, Protein A cannot be used to selectively capture human IgG derived Fab fragments from feed stock samples consisting of a mixture of human IgG Fc- and Fab fragments, due to its more prominent binding reactivity towards IgG Fc domains. In addition, Z-domain based Protein A variants (like Mab Select Sure) lack the ability to bind to human VH3 domains and as such can not be used for Fab purification.
Protein-G is a bacterial surface protein expressed by group C and G streptococci. Protein-G recognizes a common site at the interface between CH2 and CH3 domains on the Fc part of human IgG1, IgG2, IgG3 and IgG4 antibodies (Fcγ) with high affinity. In addition, Protein-G shows binding to the Fab portion of IgG antibodies through binding to the CH1 domain of IgG in combination with a CL-domain of the kappa isotype (Derrick and Wigley (1994) J. Mol. Biol. 243:906-918). Protein-G only binds to Fab from IgG1, IgG3 and IgG4, but not to Fab of IgG2 (Perosa et al (1997) Journal of Immunological Methods 203:153-155). Binding affinity towards CH1 is significantly lower compared to its epitope on the Fc part, which for instance reflects in the low flow rates which must be used to enable efficient purification of human Fab fragments (Proudfoot et al. (1992) Protein expression and purification 3:368-373). Regarding binding of Protein-G at the interface between CH2 and CH3 domains, experimental data indicate that induced fit occurs, which may explain the harsh conditions required for elution as illustrated by an elution pH at or below pH 2.5 (PROTEUS, Protein G Antibody Purification Handbook; Mini&Midi spin columns; 5 Sep. 2005; Pro-Chem, Littleton, USA). These harsh conditions may affect the conformation of the binding sites, thereby altering the immune function of purified IgG antibodies (P. Gagnon, 1996, in Purification tools for monoclonal antibodies, published by Validated Biosystems, Inc 5800N). X-ray crystallographic measurements have shown that through binding to Protein-A, the CH2 domains can be displaced longitudinally towards the CH3 domains, which finally causes partial rotation and destabilization of the carbohydrate region between the CH2 domains. The distortion interferes with subsequent protein-protein interactions that are required for the IgG to exert its effector functions. Aside from the consequences of harsh elution conditions (especially for Protein-G) on the antigen binding capabilities, these secondary effects sometimes interfere with or alter antibody effector functions and increased susceptibility of immunoglobulins to proteolysis. Loss of effector functions, caused by denaturation, altered folding and chemical modifications that arise during purification steps, are highly undesirable if the human or humanized antibodies are to be used for therapeutic purposes. In particular, reduction of intra- and inter-molecular sulphur bridges is often a problem that arises during purification and storage.
Protein L binds human Fab via VL kappa 1, 3, and 4 but does not bind to VL kappa 2 and none of the VL domains of the lambda isotype. Thus, Protein L does not bind to all human IgG derived Fab fragments and is not selective for IgG only.
As alternative to human IgG binding proteins like Protein-G, several mouse monoclonal antibodies (Mabs) have been described in literature that are capable of binding to the Fc domain of human IgG antibodies (Nelson P N, et al. Characterisation of anti-IgG monoclonal antibody A57H by epitope mapping. Biochem Soc Trans 1997; 25:373.). Some common Fc epitopes have been identified and a number of examples are: Mabs G7C, JD312 have a binding epitope on CH2, amino acids 290-KPREE-294. Mabs PNF69C, PNF110A, PNF211C, have a binding epitope on CH2-CH3, AA: 338-KAKGQPR-344. Mab A57H shows binding epitope on CH3, AA 380-EWESNGQPE-388. A problem associated with the use of mouse Mabs, or Mabs from other non-human species, is the release of Mabs from the matrix which causes contamination in the purified preparations that is difficult to remove. Furthermore, Mabs and functional fragments thereof (Fab, Fab2) are easily denatured and S—S bridges, necessary for the correct the 3D structure of the molecule and the alignment of the heavy and light chains, are easily disrupted, in particular under harsh elution conditions that are oftentimes required for release of column bound human IgG's. Due to the vulnerability of the Mab-based affinity ligands the capacity of the column is rapidly reduced, and columns have a very limited re-use capacity after elution and are unsuitable for continuous operation.
Instead of (sc)Fv fragments as described in EP-A-434317, antibody fragments derived from antibodies naturally devoid of light chains (VHH) as described in WO2006/059904 can also be used to generate immunosorbent materials for the purification of human IgG antibodies. Advantage of use of these VHH fragments are that they are single domain peptides, which are exceptionally stable even at higher temperatures. Furthermore, VHH's, are small and easily produced in cost-efficient host organisms such as Saccharomyces cerevisiae. In addition, due to the sequence similarity between these VHH fragments and the human VH3 domain family, immunogenecity is expected to be very low compared to bacterial surface proteins like Protein-A and G. These antibodies are described in more detail in EP-A-656946.
The amino acid sequences as described in WO2006/059904 relate to VHH fragments that bind to the light chain of human antibodies of either the kappa or lambda isotype, and as such do not enable selective purification of antibodies and/or Fab fragments of the IgG isotype only. In addition, the excess of light chains present in the supernatant of e.g. cell lines expressing human IgG antibodies and/or Fab fragments thereof, will also be captured by said ligands. Synthetic ligands such as Fabsorbent™ F1P HF (ProMetic BioSciences Ltd, Cambridge, UK) have the same drawbacks.
The amino acid sequences as described in WO2009/011572 relate to VHH fragments that bind to the Fc part of human IgG. As such they do not allow purification of fragments of human IgG that do not comprise the Fc domain, such as Fab of F(ab′)2 fragments of human IgG.
Carredano et al. describe the development of synthetic affinity ligands for human Fab purification, binding to a conserved cavity common to all antibodies of the IgG-kappa type (a small pocket formed between the IgG CH1 and the CL-kappa domain) (Carredano et al. (2004) Protein Science 13:1476-1488). Although the IgG-CH1 is considered to be the most conserved region among different IgG Fab fragments, Carredano et al. concluded this domain to be less suitable for generating good binding affinity ligands, because of its flat structure (as also reported in Derrick et al. supra). Rather, the above cavity was selected, hence accepting a lack of reactivity towards human Fab fragments comprising a light chain of the lambda isotype.
Thus, binding agents known in the art have been described that can be used for the purification of human IgG antibodies and/or Fab fragments thereof. However, none of these binding agents (on its own) can provide a generic purification process for all human IgG antibodies and/or Fab fragments thereof. Thus, there is a need in the art for new means and methods that allow a generic approach for the capture and/or purification of human or humanized IgG1, IgG2, IgG3 and IgG4 and/or Fab fragments thereof.